In vitro and in vivo acute toxicity of an artificial butter flavoring

ABSTRACT

Flavorings used in cookies, electronic cigarettes, popcorn, and breads contain approximately 30 chemical compounds, which makes it difficult to determine and correlate signs and symptoms of acute, subacute or chronic toxicity. The aim of this study was to characterize a butter flavoring chemically and subsequently examine the in vitro and in vivo toxicological profile using cellular techniques, invertebrates, and lab mammals. For the first time, the ethyl butanoate was found as the main compound of a butter flavoring (97.75%) and 24 h-toxicity assay employing Artemia salina larvae revealed a linear effect and LC₅₀ value of 14.7 (13.7–15.7) mg/ml (R² = 0.9448). Previous reports about higher oral doses of ethyl butanoate were not found. Observational screening with doses between 150–1000 mg/kg by gavage displayed increased amount of defecation, palpebral ptosis, and grip strength reduction, predominantly at higher doses. The flavoring also produced clinical signs of toxicity and diazepam-like behavioral changes in mice, including loss of motor coordination, muscle relaxation, increase of locomotor activity and intestinal motility, and induction of diarrhea, with deaths occurring after 48 h exposure. This substance fits into category 3 of the Globally Harmonized System. Data demonstrated that butter flavoring altered the emotional state in Swiss mice and disrupted intestinal motility, which may be a result of neurochemical changes or direct lesions in the central/peripheral nervous systems.

KEYWORDS:

• Ethyl butanoate
• flavoring agents
• food additives
• intestinal motility
• physiological changes

Acknowledgments

Paulo Michel Pinheiro Ferreira and Nárcia Mariana Fonseca Nunes are grateful to the public Brazilian agencies “Conselho Nacional de Desenvolvimento Científico e Tecnológico” [CNPq (#304803/2022-7)] and “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” [CAPES (Finance code 001)] for their personal scholarship, respectively. We also thank Leane Brunelle dos Santos Alves for technical assistance and the Postgraduate Program in Pharmaceutical Sciences (PPGCF, Teresina, Piauí, Brazil) for structural support.

Disclosure statement

No potential conflict of interest was reported by the authors.

Ethics approval

All protocols involving animals were approved by the Ethical Committee on Animal Experimentation at UFPI (CEUA #0256/2016) and followed Brazilian (Sociedade Brasileira de Ciência em Animais de Laboratório - SBCAL) and International (Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes) rules on the care and use of experimental animals. The manuscript is also in line with the Recommendations for the Conduct, Reporting, Editing and Publication of Scholarly Work in Medical Journals.

Submission declaration and verification

The work described has not been previously published or submitted for publication elsewhere. The publication is approved by all authors.

Additional information

Funding

The work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico [304803/2022-7].